Candidate: Dr. Matthew J. Sikora began his research career during his undergraduate and graduate training in pharmacology at the University of Michigan. During this time, Dr. Sikora developed an interest in translational research that moves laboratory findings to clinical application. Also, he developed a passion for cancer research, and committed to a career in breast cancer research. During his post-doctoral training at the University of Pittsburgh, Dr. Sikora began to study endocrine response and resistance in invasive lobular carcinoma (ILC), a poorly understood subtype of breast cancer. This research has allowed Dr. Sikora to develop his strengths in pharmacology and endocrine therapy, while also exploring his interest in individualized medicine and developing translational research projects. Dr. Sikora has been working toward the goal of becoming a principle investigator at a top-tier academic institution and comprehensive cancer center, with the ultimate goal of translating his research progress to improve cancer therapy and patient outcomes. Training: This research project includes important training aspects that will both augment Dr. Sikora's current research strengths and allow him to differentiate himself from his mentor. The overall focus of the research project (described below) will provide Dr. Sikora with extensive experience in elucidating signal transduction pathways, which has not previously been a component of his training. This experience is critical in understanding and targeting mechanisms of therapeutic resistance. Additionally, his focus on Wnt signaling is a new area of research for his current mentor, Dr. Steffi Oesterreich. A co-mentorship with Dr. Caroline Alexander, an expert in Wnt signaling in the mammary gland, will provide a unique direction for Dr. Sikora's independent research career. This project will also utilize novel model systems that will allow Dr. Sikora to rapidly test therapeutic strategies directly in patient tumor tissue. Throgh collaboration with Dr. Karen Knudsen, Dr. Sikora will develop the ex vivo tissue culture model to assess the function of Wnt signaling inhibitors in patient-derived ILC tissues. The training in bot signal transduction and novel model systems, in conjunction with Dr. Sikora's unique background in pharmacology and endocrine therapies, will provide Dr. Sikora with a unique niche in breast cancer research that will allow him to impact patient care. Research: The overall hypothesis of this project is that invasive lobular carcinoma cells utilize unique signaling pathways driven by WNT4 that represent putative targets to modulate endocrine response in ILC. This hypothesis will be tested in three aims: Aim 1: Identify the WNT4 receptor complex that regulates estrogen-induced growth in ILC cells. Wnt ligands bind and activate frizzled (FZD) receptors which subsequently activate dishevelled (DVL) proteins to initiate signaling; DVL proteins are also subject to direct inhibition. To identify the WNT4 receptor and associated proteins, we will use novel immunoprecipitation methods (IFAST) to identify protein-protein interactions among WNT4 and receptor proteins. This will couple with siRNA and small molecule inhibitors to identify receptor complex proteins necessary for estrogen-induced growth. Receptor complex activity will also be assessed in ex vivo cultures of cell line xenograft tumors as a novel 3D model of ILC. Aim 2: Identify the non-canonical WNT4 signaling cascade in ILC cells. A defining feature of ILC is loss of E-cadherin; uniquely in ILC cells, this also causes ?-catenin protein loss and dysfunction of canonical Wnt signaling. WNT4 likely utilizes novel non-canonical pathways in ILC. We will examine WNT4-mediated activation of signal transduction cascades using protein arrays. Additionally, we will define the specific transcriptional program downstream of WNT4 signaling via CRISPR-mediated knockout of the WNT4 gene and the ER binding site at the WNT4 locus. The role of non-canonical signaling in regulating ILC cell viability and proliferation will also be assessed in 3D models of ILC. Aim 3: Determine whether WNT4 drives estrogen-independence and endocrine resistance in ILC. This aim will initiate translation of this project toward clinical application. To examine the role of WNT4 signaling in endocrine resistance, we will use siRNA and WNT4 pathway inhibitors to target novel estrogen independent variants of ILC cell lines. Further, WNT4 pathway gene expression will be measured in ILC patient specimens with long-term follow-up data. Ex vivo cultures of patient-derived ILC tumor tissue will be used to identify therapeutic strategies that can effectively inhibit WNT4 signaling and improve endocrine response for ILC patients. ILC represents an understudied patient population, ideal for individualized therapies to improve patient outcomes. We hypothesize that targeting WNT4 and downstream signaling pathways will improve endocrine response in ILC cells. This project will elucidate previously uncharacterized pathways, and will identify putative targets for therapy to prevent or reverse endocrine resistance in ILC. By testing hypotheses in novel models, archival patient samples, and patient-derived tissues, this project will rapidly translate results to clinical application to ultimately improve ILC patient outcomes.